The recent economic advances and lifestyle changes have been accompanied by great changes in dietary habit. Particularly, busy people of today are overweight and obese due to high-calorie diets and insufficient exercise. The World Health Organization (WHO) has reported that more than one billion adults are overweight worldwide, among them over three million are clinically diagnosed with severe obesity, and 25,000 people die of overweight- or obesity-related diseases every year (World Health Organization, Global Strategy on Diet, Physical Activity and Health, 2004).
Overweight and obesity increase blood pressure and blood cholesterol level, thereby increasing the risk of various adult diseases including heart disease, diabetes, arthritis, etc. Further, overweight and obesity are one of the main causes for inducing adult diseases such as arteriosclerosis, hypertension, hyperlipidemia and heart disease in children and adolescents.
Obesity is now recognized as a serious disease prevalent over the world and is a cause of various diseases. However, it is not readily curable. Medical specialists have the conception that obesity is not merely a problem of self-control but is a complicated disease which is closely related to appetite control and energy metabolism. Obesity results from excessive energy intake relative to energy consumption. At the individual level, a combination of excessive food energy intake and a lack of physical activity is thought to explain most cases of obesity. Therefore, in the long-term, there is an urgent need to develop a medicine which is effective and safe in the prevention and treatment of obesity when used in combination with diet therapy and physical activity.
In recent medicinal research on obesity, keen attention has been paid to oxyntomodulin, which is derived from pre-glucagon and can bind to both the GLP-1 and glucagon receptors. The development of an anti-obesity drug based on such a dual function of oxyntomodulin has been actively studied.
Glucagon-like peptide-1 (hereinafter, referred to as ‘GLP-1’) is a drug under development for hyperglycemia in diabetic patients and possesses several physiological functions of increasing insulin synthesis and secretion, decreasing glucagon secretion, inhibiting gastric emptying, enhancing the use of glucose and decreasing food intake. Also, exendin-4 which is secreted by lizard venom and shows about 50% homology with GLP-1, is known to alleviate hyperglycemia in diabetes patients by activating the GLP-1 receptor.
Glucagon is secreted by the pancreas when blood glucose levels fall too low due to, for example, drug treatment, disease, hormones or enzyme deficiency. Glucagon signals the liver to break down glycogen to glucose and release more glucose into the blood stream, which results in raising blood glucose level.
Acting as an agonist to both the GLP-1 receptor and the glucagon receptor, oxyntomodulin has the functions of decreasing food intake like GLP-1 and digesting glycogen like glucagon, and thus is a potential anti-obesity agent.
As an oxyntomodulin derivative, there are a dual agonist peptide (Merck) in phase 1 clinical trials, and ZP2929 (Zealand, WO 2008/152403A1) in pre-clinical trials. The dual agonist peptide (Merck) is an oxyntomudulin derivative whose resistance to dipeptidyl peptidase-IV (hereinafter, referred to as ‘DPP-IV’) is increased by substituting the 2nd amino acid L-Ser with D-Ser, and blood half-life is prolonged by conjugating a cholesterol moiety to the C-terminal end thereof. ZP2929 (Zealand) is an oxyntomodulin derivative which includes substitutions of the 2nd amino acid L-Ser with D-Ser to increase resistance to DPP-IV, the 17th amino acid Arg with Ala to increase resistance to proteases, the 27th amino acid Met with Lys to increase oxidative stability, and the 20th and 24th amino acids Gln and the 28th amino acid Asn with Asp, Aln and Ser, respectively, to increase deamidation stability.
The ability of oxyntomodulin to act on both the GLP-1 receptor and the glucagon receptor offers promise in the development of a potential anti-obesity agent. However, the problem in the therapeutic use of oxyntomudulin is the extremely short half-life (8˜12 min). The blood half-life of the dual agonist oxyntomodulin, although longer than that of native oxyntomodulin, still remains as short as 7 hours in vivo. Further, its administration dose is as high as several mg/kg. Thus, oxyntomodulin or its derivatives currently used suffer from the disadvantage that it has to be administered daily at a high dose due to short half-life and low efficacy. In case of protein drugs comprising such a peptide as an effective ingredient, it is necessary to administer the drug frequently through an injection so as to maintain the proper blood levels and titers, which is very painful to the patients. Therefore, the maintenance of high blood levels and proper efficacy is a prerequisite for the development of effective anti-obesity drugs.
Meanwhile, many attempts have been made to maximize drug efficacy by improving blood stability of a protein drug and maintaining high blood level thereof over a long period. In order for this, a method of chemically modifying the surface of a drug with a polymer such as polyethylene glycol (PEG) has been developed. However, the method has problems in that the higher the molecular weight of PEG, the lower its reactivity with a target protein, leading to a decrease in pegylation yield. Alternatively, International Patent Publication No. WO 02/46227 describes a fusion protein prepared by coupling a target protein with human blood albumin or an immunoglobulin region (Fc) via genetic recombination. U.S. Pat. No. 6,756,480 describes an Fc fusion protein prepared by coupling a parathyroid hormone (PTH) and an analog thereof with an Fc region. These methods can address the problems such as low pegylation yield and non-specificity, but they still have problems in that the effect of increasing the blood half-life is not noticeable as expected, and occasionally the titers are also low. In order to maximize the effect of increasing blood half-life, various kinds of peptide linkers are used, but an immune response may be possibly caused. Further, if a peptide having a disulfide bond is used, there is a high probability of misfolding.
In order to solve these problems, the present inventors have suggested a long-acting protein conjugate in which a physiologically active polypeptide and an immunoglobulin Fc fragment are covalently linked to each other via a non-peptide linker, thereby improving blood stability while maintaining a loss of drug activity (Korean Patent No. 10-0725315). Particularly, a long-acting exendin-4 conjugate in which exendin-4 or its derivative is covalently linked to an immunoglobulin Fc fragment via a non-peptide linker showed prolonged in vivo durability (Korean Patent Laid-Open Publication No. 10-2008-0064750).
Further, the present inventors have tried to apply the long-acting exendin-4 conjugate to the development of anti-obesity drugs with improved in vivo durability and stability. In this regard, the long-acting exendin-4 conjugate (once a week) was administered in combination with native glucagon (once a day) to simultaneously stimulate both the GLP-1 receptor and the glucagon receptor. When administered in combination with native glucagon, the long-acting exendin-4 brought about exceptional weight loss, compared to when administered alone. However, said co-administration caused severe fluctuation in blood glucose level, showing toxicity in test animals.
The present inventors have therefore endeavored to develop a method of increasing blood half-life of glucagon while maintaining in vivo activity thereof, and contrived a novel long-acting glucagon conjugate in which glucagon or its derivative is covalently linked to a polymer carrier such as an immunoglobulin Fc fragment via a non-peptide linker. The long-acting glucagon conjugate of the present invention shows improved in vivo durability and stability. Thus, when used in combination with an anti-obesity drug, the long-acting glucagon conjugate of the present invention can efficiently reduce body weight and food intake at a significantly low dose. Further, such co-administration exhibits better drug compliance without causing severe fluctuation in blood glucose level. Accordingly, the long-acting glucagon conjugate of the present invention can be effectively used for the prevention and treatment of obesity.